Protocol for photo-controlling the assembly of cyclic peptide nanotubes in solution and inside microfluidic droplets

Summary

In this protocol, we describe how to perform the photo-isomerization of cyclic peptides containing an unsaturated β-amino acid. This process triggers the formation or disassembly of cyclic peptide nanotubes under appropriate light irradiation. Specifically, we start by describing the solid-phase synthesis of the cyclic peptide component. We also present a technique for performing isomerization studies in solution and how to extend it to microfluidic aqueous droplets.

For complete details on the use and execution of this protocol, please refer to Vilela-Picos et al.¹

Graphical abstract

Highlights

• •Step-by-step details for the solid-phase synthesis of cyclic peptides
• •Instructions for photo-isomerization of cyclic peptides
• •Guidance on preparation of a PDMS microfluidic device
• •Steps for studying self-assembly in confined spaces and fusion of microfluidic droplets

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

In this protocol, we describe how to perform the photo-isomerization of cyclic peptides containing an unsaturated β-amino acid. This process triggers the formation or disassembly of cyclic peptide nanotubes under appropriate light irradiation. Specifically, we start by describing the solid-phase synthesis of the cyclic peptide component. We also present a technique for performing isomerization studies in solution and how to extend it to microfluidic aqueous droplets.

Before you begin

This protocol describes how to study the photo-isomerization and subsequent self-assembly of cyclic peptide nanotubes both in solution and in microfluidic experiments. Self-assembling cyclic peptide nanotubes (SCPNs) are formed by the ordered stacking of cyclic peptide units driven by the formation of hydrogen bonds between neighboring peptide components.^2,3,4 In this context, we have previously implemented this protocol for inducing the assembly or disassembly of this type of supramolecular polymers by light irradiation.¹ This process is based on the Z-/E- isomerization of an unsaturated β-alanine derivative.^5,6,7,8 The Z-isomer peptide exhibits a folded-conformation that prevents it from stacking, while the E-isomer adopts the flat conformation necessary for the assembly. The process can also be carried out in confined environments with a good spatiotemporal control and represents a complementary approach to our previous work in which a UV light-mediated anthracene [4 + 4] cycloaddition transformation bent SCPNs.⁹

Droplet-based microfluidic experiments represent a powerful tool for studying supramolecular systems.^10,11,12,13 Not only do they provide greater control than bulk experiments, but they also allow evaluation in confined spaces of the behavior of fibril-like structures as mimetics of natural cytoskeleton. As a result, they are an ideal complement to experiments in solution. In this particular case, they enable to study the spatiotemporally controlled supramolecular polymerization of CPs within aqueous droplets.^1,14 Through the application of the microfluidic protocol described here, we have discovered the fusogenic properties under physiological conditions of nanotubes formed from the aforementioned E-isomer, due to their ability to cause droplets fusion.¹

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Fmoc-Rink amide AM resin	Iris Biotech	CAS 183599-10-2
Piperidine	Thermo Fisher Scientific	CAS 110-89-4
Fmoc-Glu-OAll	Carbolution	CAS 144120-54-7
Fmoc-D-His(Trt)-OH	Carbolution	CAS 135610-90-1
Fmoc-Ser(tBu)-OH	Carbolution	CAS 71989-33-8
Fmoc-D-Lys(Mtt)-OH	Carbolution	CAS 198544-94-4
Fmoc-Ala-OH	Carbolution	CAS 35661-39-3
Fmoc-D-Ser(tBu)-OH	Carbolution	CAS 128107-47-1
Fmoc-His(Trt)-OH	Carbolution	CAS 109425-51-6
Fmoc-D-Gln(Trt)-OH	Carbolution	CAS 200623-62-7
Fmoc-β-Ala-OH	Sigma-Aldrich	CAS 35737-10-1
Fmoc-PΔβAla-OH	Marafon et al.5	Marafon et al.5
N-HBTU (N-[(1H-Benzotriazol-1-yl)-(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate-N-oxide)	Carbolution	CAS 94790-37-1
N-HATU (N-[(Dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methyl methanaminium hexafluorophosphate N-oxide)	Carbolution	CAS 148893-10-1
DIEA (N,N-diisopropylethylamine)	Sigma-Aldrich	CAS 7087-68-5
Palladium (II) acetate	Sigma-Aldrich	CAS 3375-31-3
Triphenylphosphine	Sigma-Aldrich	CAS 603-35-0
Phenylsilane	TCI Chemicals	CAS 694-53-1
NMM (4-methylmorpholine)	Sigma-Aldrich	CAS 109-02-4
PyAOP (7-azabenzotriazol-1 yloxy)tripyrrolidinophosphonium hexafluorophosphate)	Carbolution	CAS 156311-83-0
Sodium diethyldithiocarbamate	Sigma-Aldrich	CAS 20624-25-3
Lithium chloride	Sigma-Aldrich	CAS 7447-41-8
HFIP (1,1,1,3,3,3-hexafluoro-2-propanol)	Fluorochem	CAS 920-66-1
TFE (2,2,2-trifluoroethanol)	Fisher Scientific	CAS 75-89-8
TIS (triisopropylsilane)	Sigma-Aldrich	CAS 6485-79-6
1-pyreneacetic acid	Sigma-Aldrich	CAS 64709-55-3
TFA (trifluoroacetic acid)	Fisher Scientific	CAS 76-05-1
Thioanisole (methyl phenyl sulfide; MPS)	Sigma-Aldrich	CAS 100-68-5
DMF (N,N-dimethylformamide)	Fisher Scientific	CAS 68-12-2
DCM (dichloromethane)	Fisher Scientific	CAS 75-09-2
Diethyl ether	Fisher Scientific	CAS 60-29-7
ACN (acetonitrile)	Fisher Scientific	CAS 75-05-8
MilliQ H2O	Millipore Q-POD	CAS 7732-18-5
HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)	TCI Chemicals	CAS 7365-45-9
Boric acid	Sigma-Aldrich	CAS 10043-35-3
NaOH	Fisher Scientific	CAS 1310-73-2
HCl	Fisher Scientific	CAS 7647-01-0
ABA (4-aminobenzoic acid)	Sigma-Aldrich	CAS 150-13-0
SYLGARD 184 Silicone Elastomer Kit	Dow Corning	Material number 1673921
Trichloro(1H,1H,2H,2H-perfluorooctyl)silane	Sigma-Aldrich	CAS 78560-45-9
HFE-7500 3M Novec	Fluorochem	CAS 297730-93-9
Pico-Surf (5% in Novec 7500)	Sphere Fluidics	Product code F006
CaCl2	Fluorochem	CAS 10043-52-4
Software and algorithms
GraphPad Prism	GraphPad	https://www.graphpad.com/
ImageJ	ImageJ	https://imagej.net/
ChemDraw Professional 22.0	PerkinElmer	https://www.perkinelmer.com/category/chemdraw
Photron FASTCAM Viewer (PFV)	Photron	https://photron.com/photron-support
Other
Poly-Prep chromatography columns	Bio-Rad	731-1550
250 mL three-neck round bottom flask	Scharlab	073-314149
10 mL two-neck round bottom flask	Sigma-Aldrich	SYNF411110
Rotatory shaker	Lan Technics	LAN-RD
50 mL centrifuge tubes with screw caps	VWR	525-1099
2 mL Eppendorf Safe-Lock tubes	Eppendorf	0030120094
Thermo-shaker	Biosan	TS-100C
HPLC Hitachi D-7000 with a Phenomenex Luna 5 μm-C18 column	Hitachi and Phenomenex	D-7000
uHPLC-MS Agilent Technologies 1260 Infinity II with a 6120 Quadrupole LC-MS and an Agilent SB-C18 column	Agilent Technologies	1260 Infinity II
Lyophilizer	Buchi	Lyovapor L-300
Balloon	Sigma-Aldrich	Z154989
Needle 0.80 × 50 mm	Braun	4665503
Hellma quartz cuvettes with 2 mm pathlength	Hellma Analytics	Z800120
Stand base	Labbox	RTSP-310-001
Glass funnel	Labbox	FUS3-100-006
Clamp “Triplex”	Scharlab	0197000160
Double bosshead	Scharlab	0191000069
UV lamp 290–320 nm	Phillips	TL 20W/12 RS
UV lamp 254 nm	Phillips	TUV 15W/G15 T8
Screw vial for chromatography	Labbox	SVSC-C20-1K0
Inserts for screw neck vials	VWR	548-1103
Master mold	Méndez-Ardoy et al.14	Méndez-Ardoy et al.14
Desiccator	Normax	4.4906524
Oven	Memmert	UNB 300
Sharp razor	Scharlab	0000CUTTER
1 mm diameter biopsy punch	KAI Medical	109199
Glass slides	Fisher Scientific	12383118
Plasma cleaner	Fischione	1020
Sonicator	Branson Ultrasonics	2800
1 mL syringes	Braun	9166017V
Bore polythene tubing (Portex, 0.38 × 0.35 mm).	Scientific Laboratory Supplies	tub3656
Two-channel microfluidics syringe pump	syringepump.com	NE-4002X
Inverted microscope with a high-speed camera setup	Nikon and Photron	Eclipse Ti2 and Fastcam Mini UX
Epifluorescence microscope	Olympus	BX51
Spinning disk confocal setup on an inverted microscope	Andor and Nikon	DragonFly and Eclipse Ti-E

Materials and equipment

Chemicals must be stored according to the manufacturer’s specifications. All solvents used are HPLC or synthesis grade. To obtain dry DCM, it can be distilled over CaH₂. The mixture of HFIP, TFE and TIS in DCM (20:10:5:65) and the mixture of MPS, H₂O, DCM in TFA (2.5:2.5:5:90) should be freshly prepared.

Mtt deprotection solution

Reagent	Final concentration	Amount
HFIP (1,1,1,3,3,3-Hexafluoro-2-propanol)	20% (v/v)	800 μL
TFE (2,2,2-Trifluoroethanol)	10% (v/v)	400 μL
TIS (Triisopropylsilane)	5% (v/v)	200 μL
DCM (Dichloromethane)	65% (v/v)	2,6 mL
Total	N/A	4 mL

The solution should be freshly prepared at the time of use.

TFA cleavage and deprotection solution

Reagent	Final concentration	Amount
TFA (Trifluoroacetic acid)	90% (v/v)	3,6 mL
Thioanisole (methyl phenyl sulfide; MPS)	2.5% (v/v)	100 μL
H2O	2.5% (v/v)	100 μL
DCM (Dichloromethane)	5% (v/v)	200 μL
Total	N/A	4 mL

The solution should be freshly prepared at the time of use.

Step-by-step method details

Cyclic peptide synthesis

Timing: 2 weeks

This section describes the synthesis of the cyclic peptide structures (CP2 and ^ZCP1, Figure 1) required for the development of the protocol. First, CP2 is synthesized by solid-phase synthesis using an Fmoc/^tBu methodology.^15,16 After reverse-phase HPLC purification, CP2 containing a furan cycloadduct to mask the olefin moiety is deprotected to obtain ^ZCP1 by heating at 80°C a sample solubilized in H₂O/ACN (1:1).⁵

• 1.
  Synthesis of the linear peptide.

  • a.
    Weigh the Rink Amide resin in a reaction vessel (400 mg, 256 μmol, Loading: 0.64 mmol/g) (Figure 1B).
  • b.
    Add 4 mL of DCM and stir in a rotatory shaker for 30 min to swell the resin (Figure 1C).

    • i.
      Then, filter the resin by applying vacuum (Figure 1D).

      Note: Make sure that all resin is being shaken. A suggested agitation speed to all steps is 80 rpm.

  • c.
    Treat the resin with 4 mL of piperidine/DMF (1:3) and stir bubbling argon for 20 min to remove the Fmoc protecting group (Figure 1D).
  • d.
    Filter the resin and wash it with DMF (3 × 4 mL, 3 min) and DCM (3 × 4 mL, 3 min) (Figure 1D).
  • e.
    In a vial, dissolve the Fmoc-amino acid to be coupled (3 equiv) and N-HBTU (3 equiv) in DMF. Next, add DIEA (6 equiv) and pour the solution into the reaction vessel with the resin.

    • i.
      Shake for 40 min in a rotatory shaker (Figure 1C).
    • ii.
      Then, wash with DMF (3 × 4 mL, 3 min) (Figure 1D).
  • f.
    Repeat steps 1c-e until the linear peptide synthesis is completed.

    Note: The first coupling must be done using the side chain of Fmoc-Glu-OAll (1), so that the cyclization can be performed on the resin in the step 2. The last one to be coupled is the photosensitive amino acid protected as a furan cycloadduct (2), whose deprotection is carried out in step 5.

    Note: To check the proper progress of the synthesis at a given time, a small test-cleavage and uHPLC-MS analysis can be performed. To do this, a small amount of resin (2–3 mg) is added to an Eppendorf tube and treated with a mixture of MPS, H₂O, DCM in TFA (2.5:2.5:5:90, 100 μL) for 2 h. After this time, Et₂O is added (900 μL), the Eppendorf tube is centrifuged, decanted and the pellet is dissolved in H₂O+0.1% TFA and analyzed by uHPLC-MS.

• 2.
  Allyl deprotection and cyclization.

  • a.
    Add to the resin a mixture of Pd(OAc)₂ (0.25 equiv), PPh₃ (1.25 equiv), N-methylmorpholine (6 equiv), and phenylsilane (6 equiv) in dry DCM (4 mL) to remove the allyl protecting group.
  • b.
    Shake the mixture for 2 h in a rotatory shaker (Figure 1C).

    Note: During this process, overpressure may form inside the reaction vessel.

  • c.
    Filter the resin and wash it with DCM (3 × 4 mL), DMF (3 × 4 mL), sodium diethyldithiocarbamate in DMF (0.5% v/v, 2 × 4 mL, 15 min), DMF (2 × 4 mL), DIEA in DMF (10% v/v, 2 × 4 mL) and DMF (3 × 4 mL) (Figure 1D).
  • d.
    Remove the N-terminal Fmoc group by treating the resin with piperidine in DMF (1:3, 4 mL) for 30 min (Figure 1D).
  • e.
    Filter the resin and wash with DMF (3 × 4 mL), DCM (3 × 4 mL), DIEA in DMF (5% v/v, 3 × 4 mL), LiCl in DMF (0.8 M, 3 × 4 mL) and DMF (3 × 4 mL) (Figure 1D).
  • f.
    Treat the resin with a solution of PyAOP (3 equiv) and DIEA (6 equiv) in DMF (4 mL) for 2 h (Figure 1C).
  • g.
    Filter the resin and wash with DMF (3 × 4 mL) and DCM (3 × 4 mL) (Figure 1D).
• 3.
  Mtt deprotection and side chain functionalization.

  • a.
    Treat the resin with a freshly prepared mixture of HFIP, TFE and TIS in DCM (20:10:5:65; 4 mL) for 2 h to deprotect the Mtt protecting group on the lysine side chain (Figure 1C).

    • i.
      Repeat this process three times.
    • ii.
      After each cycle, wash with DCM (3 × 3 mL) (Figure 1D).
  • b.
    Add to the resin a solution of 1-pyreneacetic acid (3 equiv), N-HATU (3 equiv) and DIEA (6 equiv) in DMF (4 mL) and stir for 40 min (Figure 1C).
  • c.
    Wash the resin with DMF (3 × 4 mL) and DCM (3 × 4 mL) (Figure 1D).
• 4.
  Peptide cleavage and purification.

  • a.
    Treat the resin with a mixture of MPS, H₂O, DCM in TFA (2.5:2.5:5:90, 4 mL) for 2 h (Figure 1C).

    Note: Triisopropylsilane (TIS) must not be used because it can cause reduction of the double bond of compound 2 (Figure 1A).

  • b.
    Filter the solution into a two-neck round bottom flask (Figure 1E).
  • c.
    Concentrate the resulting solution by bubbling argon until obtaining 1–2 mL (Figure 1F).
  • d.
    Add the mixture dropwise to cold Et₂O (40 mL of Et₂O for 1 mL of TFA) (Figure 1G).
  • e.
    Collect the precipitate by centrifugation (4000 rpm × 10 min). The supernatant is discarded (Figure 1H).
  • f.
    Wash the solid with cold Et₂O and centrifugate again (4000 rpm × 10 min).
  • g.
    Dry the solid.
  • h.
    Dissolve the solid in MilliQ-H₂O with 0.1% TFA and purified by reverse-phase high-performance liquid chromatography (RP-HPLC) to obtain CP2. [HPLC conditions: Phenomenex Luna C18 (2) 100 Å column with a gradient of H₂O (0.1% TFA)/ACN (0.1% TFA), 80:20 → 80:20 (5 min); 80:20 → 50:50 (40 min), R_t = 25.6 min].
  • i.
    Collect the pure fractions in a round bottom flask, evaporate acetonitrile at the rotary evaporator, and lyophilized to afford the pure CP2 as a white solid.
• 5.
  Furan cycloadduct deprotection.

  • a.
    Prepare a solution of CP2 at 1.5 mM in H₂O/ACN (1:1) in Eppendorf Tubes.
  • b.
    Shake it at 80°C during 24 h in a thermo-shaker. Cover the reaction with aluminum foil (Figure 1I).
  • c.
    Purify the resulting mixture by reverse-phase high-performance liquid chromatography (RP-HPLC) to obtain ^ZCP1. [HPLC conditions: Phenomenex Luna C18 (2) 100 Å column with a gradient of H₂O (0.1% TFA)/ACN (0.1% TFA), 80:20 → 80:20 (5 min); 80:20 → 50:50 (40 min), R_t = 25.8 min].
  • d.
    Combine the pure fractions in a round bottom flask, evaporate acetonitrile at the rotary evaporator, and lyophilized to obtain the pure ^ZCP1 as a white solid.

Figure 1.

Solid-phase synthesis of CP2 and deprotection to obtain ^ZCP1

(A) Synthetic scheme to obtain CP2 and ^ZCP1.

(B) Rink amide resin inside a solid-phase reaction vessel.

(C) Rotatory shaker used to stir the resin. The reaction vessel is introduced inside a Falcon tube.

(D) Argon bubbling and vacuum filtration.

(E) Filtration after cleavage with a mixture of Thioanisole (MPS), H₂O, DCM in TFA (2.5:2.5:5:90).

(F) Concentration of the cleavage mixture by bubbling of argon.

(G) Peptide precipitation upon addition to cold Et₂O.

(H) Crude peptide after precipitation and centrifugation.

(I) Stirring and heating at 80°C to perform the conversion of CP2 to ^ZCP1.

Light-isomerization of CPs in solution

Timing: 4 h

This section describes the sample preparation and light-induced isomerization of CPs in solution. The isomerization of ^ZCP1 into ^ECP1 is obtained by irradiation at 290–320 nm, while the reverse process requires irradiation at 254 nm (Figure 2A).

• 6.
  Sample preparation.

  • a.
    Prepare a solution of ^ZCP1 or ^ECP1 (200 μM) at the desired pH.

    Note: To adjust the pH of the medium, NaOH (0.1 M), HCl (0.1 M), or a buffer such as HEPES (10 mM, pH 7.0) or Boric Acid (10 mM, pH 9.0) can be used.

  • b.
    Deoxygenate the solution by bubbling argon for 5 min with a balloon (Figure 2B).
  • c.
    Add the sample to a quartz cuvette under a micro-hood flushing argon (Figure 2C).
  • d.
    Let the sample equilibrate for 5 min.
• 7.
  Irradiation.

  • a.
    Set a distance of 5 cm between the lamp and the cuvette containing the sample.
  • b.
    Irradiate the sample with a 290–320 nm lamp for the ^ZCP1 solution or with a 254 nm lamp for the ^ECP1 solution (Figure 2D).

    • i.
      For subsequent quantification of the isomerization, collect an aliquot (20 μL) of the irradiated solution at each specific time point to be analyzed.
• 8.
  uHPLC-MS analysis.

  • a.
    Dilute the irradiated aliquots (20 μL) with milli-Q water + 0.1% TFA (20 μL) and an aqueous solution of 4-aminobenzoic acid (ABA, 0.03 mg/mL) (20 μL). The ABA is used as an internal standard.
  • b.
    Add the sample to an uHPLC-MS vial.
  • c.
    Analyze the samples by uHPLC-MS. [HPLC conditions: Agilent Technologies 1260 Infinity II with a 6120 Quadrupole LC-MS using an Agilent SB-C18 column and a gradient of H₂O (0.1% TFA)/ACN (0.1% TFA), 100:0 → 80:20 (2 min); 80:20 → 50:50 (19 min), R_t (^ZCP1) = 11.6 min and R_t (^ECP1) = 11.0 min].
  • d.
    Quantify the percentage of each isomer by integrating the area of each peak in the chromatogram (Figures 2E and 2G).

Note: The graphs can be represented by using the software GraphPad Prism. A free alternative to represent the graphs could be OpenOffice Calc.

Note: The self-assembly properties of both isomers and the light-induced isomerization can be also studied by various techniques, such as Fluorescence Spectroscopy, Circular Dichroism Spectroscopy, Scanning Transmission Electron Microscopy (STEM) or Atomic Force Microscopy (AFM).

Figure 2.

Light-induced isomerization of CPs

(A) Scheme of CPs isomerization by light irradiation and ^ECP1 self-assembly.

(B) Deoxygenation of the CP starting solution by bubbling argon.

(C) Micro-hood flushing argon for the addition of the CP solution to a quartz cuvette.

(D) Sample irradiation in a quartz cuvette.

(E) Chromatograms and time-dependent conversion at pH 5.1 of ^ZCP1 into ^ECP1 by irradiation at 290–320 nm wavelength. The axes of the irradiated chromatograms were adjusted for better visualization of the peaks.

(F) STEM micrograph achieved by deposition of a ^ZCP1 solution (200 μM) in HEPES buffer (10 mM, pH 7.0) after 2 min of irradiation at 290–320 nm. Scale bar, 200 nm.

(G) Chromatograms and time-dependent conversion at pH 4.6 of ^ECP1 into ^ZCP1 by irradiation at 254 nm wavelength. The axes of the irradiated chromatograms were adjusted for better visualization of the peaks.

(H) STEM micrograph achieved by deposition of a ^ECP1 solution (25 μM) in HEPES buffer (10 mM, pH 7.0) after 6 min of irradiation at 254 nm. Scale bar, 200 nm.

Fabrication of a microfluidic device

Timing: 2 days

In this step of the protocol, we describe how to produce a PDMS microfluidic chip as the one shown in Figure 3A.

• 9.
  PDMS chips preparation (Figure 3C).

  • a.
    Thoroughly mix the elastomer and curing agent in a ratio 10:1.
  • b.
    Pour the mixture into the master mold (Figure 3B).
  • c.
    Apply vacuum to the master mold to remove all the bubbles.
  • d.
    Cure in the oven at 70°C overnight.
  • e.
    Cut the different circuits of the elastomer with a sharp razor.
  • f.
    Punch holes in the circuit with a 1 mm diameter biopsy punch.
• 10.
  Bounding of PDMS chips to glass slides (Figure 3C).

  • a.
    Treat glass slides and the previously prepared PDMS chips with oxygen plasma for 10 s.
  • b.
    Bring to contact and press each PDMS chip to a glass slide to bound them.
  • c.
    Complete the bounding by heating in the oven the resulting chips at 120°C for 1 h.
• 11.
  Silanization (Figure 3C).

  • a.
    Treat the chip channels with trichloro(1H,1H,2H,2H-perfluorooctyl)silane in HFE-7500 3M Novec (1% v/v).
  • b.
    Rinse the channels with HFE-7500 3M Novec until all channels are filled.
  • c.
    Dry the channels with a stream of argon.
  • d.
    Heat the chips in the oven at 65°C overnight.

Figure 3.

Microfluidic device preparation

(A) Illustration of the microfluidic device described in this protocol for the generation of droplets (water-in-oil). Channels dimensions: 35 × 50 μm. An aqueous CP solution (1.0 mM, milliQ water, pH 2–3, blue) is injected in I_CP, aqueous triggering solution in I_T [HEPES (20 mM, pH 7.0) or CaCl₂ (1.0 M), red] and an oil phase in I_oil (HFE 7500 3M Novec with 0.5% v/v Pico-Surf as surfactant, black). Flow rates: I_oil = 500 μL h⁻¹; I_CP = 300 μL h⁻¹; I_T = 150 μL h⁻¹. The aqueous phases are mixed in J₁ and then converge with the oil phase in J₂ for the formation of droplets.

(B) Master mold used for the preparation of the PDMS chips of this protocol.

(C) Step-by-step illustration for fabricating a PDMS microfluidic device.

Microfluidic droplets preparation

Timing: 4 h

Here we describe how to obtain microfluidic droplets (water-in-oil) using the previously prepared chips. These droplets allowed us to study the behavior of ^ZCP1 or ^ECP1 in confined spaces.

• 12.
  Droplets (water-in-oil) generation.

  • a.
    Prepare a solution of ^ZCP1 or ^ECP1 (1.0 mM, I_CP) in MilliQ water (pH 2–3) and sonicate for 60 min.
  • b.
    Prepare the oil phase (I_oil) by diluting the commercial Pico-Surf solution to 0.5% (v/v) with HFE-7500 3M Novec.
  • c.
    Prepare the triggering solution [HEPES buffer (20 mM, pH 7.0) or CaCl₂ (1.0 M), I_T].
  • d.
    Take these prepared solutions with 1 mL syringes provided with bore polythene tubing at the outlet (Portex, 0.38 × 0.35 mm) (Figure 4A).
  • e.
    Place each syringe in a pump (Figure 4B).
  • f.
    Connect each tube to the appropriate channel of the chip (Figure 4C).
  • g.
    Place an Eppendorf tube at the chip outlet (Figure 4D).
  • h.
    Pump only the oil phase into the chip to equilibrate it (I_oil = 500 μL h⁻¹).
  • i.
    Pump the aqueous solutions (I_CP = 300 μL h⁻¹ and I_T = 150 μL h⁻¹).
  • j.
    Visualize the droplets formation inside of the device by microscopy (Figure 4E; Methods video S1).
  • k.
    Collect the droplets at the outlet of the chip over HFE-7500 3M Novec containing Pico-Surf (0.5% v/v) (Figure 4D).
  • l.
    Visualize the droplets by epifluorescence or confocal microscopy (Figures 4F and 4G).

Note: Microscopy images can be processed using ImageJ software.

• 13.
  CPs isomerization inside microfluidic droplets.

  • a.
    Prepare droplets containing ^ZCP1 or ^ECP1 as described in the step 12 of this protocol.
  • b.
    Seal the droplets in a quartz cuvette and irradiate at 290–320 nm (for droplets containing ^ZCP1) or 254 nm (for droplets containing ^ECP1) according to the instructions in step 7.
  • c.
    Visualize the irradiated droplets by epifluorescence or confocal microscopy.

Figure 4.

Microfluidic droplets generation

(A) Bore polythene tubing (Portex, 0.38 × 0.35 mm) connected to a 1 mL syringe.

(B) Syringe pumps and microscope for microfluidic chip visualization.

(C) Connection of the tubing to the PDMS device.

(D) Final distribution of the PDMS chip with the tubes.

(E) Microscope visualization of the mixing of the aqueous phases and the droplets formation.

(F) Microfluidic droplets (water-in-oil) of ^ZCP1 or ^ECP1 at pH 7.0. I_CP is an aqueous solution of ^ZCP1 or ^ECP1 (1.0 mM), I_T is HEPES buffer (20 mM, pH 7.0) and I_oil is HFE 7500 3M Novec with Pico-Surf (0.5% v/v). Flow rates: I_oil = 500 μL h⁻¹; I_CP = 300 μL h⁻¹; I_T = 150 μL h⁻¹. Scale bars, 100 μm.

(G) Droplets fusion at neutral pH after leaving the microfluidic device. Scale bars, 100 μm.

Expected outcomes

This protocol can be used to control the assembly and disassembly of cyclic peptide nanotubes under light irradiation due to the different conformation of ^ZCP1 (folded) and ^ECP1 (flat). First, the solid-phase synthesis of CP2 proceeds with an estimated yield of 18%, and is followed by the deprotection of the unsaturated amino acid to obtain ^ZCP1 in a 91% yield (Figure 1). The photo-induced isomerization of CPs can be evaluated with the appropriate light irradiation. The irradiation of a ^ZCP1 sample at 290–320 nm provides ^ECP1 in a 7:3 ratio with the starting Z-isomer (Figure 2E), to provide ^ECP1 in 61% yield and recovering a 27% of ^ZCP1. Moreover, this ^ECP1 can be re-isomerized to ^ZCP1 in an almost 1:1 conversion by irradiation at 254 nm (Figure 2G). This two-isomerization process allows to control the nanotube assembly and disassembly by light irradiation (Figures 2F and 2H). This process can be complemented with supramolecular studies by Fluorescence Spectroscopy, Circular Dichroism Spectroscopy, Scanning Transmission Electron Microscopy (STEM), or Atomic Force Microscopy (AFM), among others.

The protocol also describes how to prepare a PDMS microfluidic device and how to use it to study the self-assembly of CPs inside of droplets (water-in-oil). The droplets generated with ^ZCP1 at neutral pH present quenched blue fluorescence characteristic of monomeric species, while the droplets with ^ECP1 show blue fluorescent fibril-like structures located mainly at the periphery of the droplets (Figure 4F). These peripheral accumulations endow the E-isomer with self-fusogenic properties, as evidenced by the observed droplet fusion (Figure 4G). The use of CaCl₂ reduces this fusion due to the preferential accumulation of the peptide in the core of the droplets under these high ionic strength conditions. In addition, the light-induced assembly can be also achieved inside of the microfluidic droplets.

Limitations

The steps described here represent a reproducible method for the CPs synthesis, the isomerization studies, and the evaluation of the self-assembly properties in microfluidic droplets. This protocol should also be applicable for the study of other CPs designs. However, the isomerization conversion might vary depending on the ring size and the sequence of amino acids. Furthermore, these structural changes could make more difficult to separate the two isomers by HPLC. Microfluidic experiments are also limited by the master mold available for creating the PMDS chips.

Troubleshooting

Problem 1

Allyl deprotection, cyclization or Mtt deprotection is not completed in the time established in the protocol (step 2 and 3).

Potential solution

Shake the resin using the same conditions for a longer time until the reaction is fully completed. Another option is to drain and wash the resin and repeat the reaction.

Problem 2

Formation of overpressure inside the reaction vessel during allyl deprotection (step 2b).

Potential solution

Divide the total amount of resin into two different reaction vessels to reduce the formation of overpressure. Opening the cartridge after 1 min of agitation may also help to eliminate the initial overpressure.

Problem 3

Minor formation of an oxidized derivative is observed by uHPLC-MS during CPs isomerization (steps 6, 7, 8).

Potential solution

Ensure that the sample is well deoxygenated and that the irradiation is carried out in an inert atmosphere to avoid the formation of oxidation derivatives.

Problem 4

In the isomerization steps, uHPLC-MS shows a reduction of peptide signals at neutral or basic pH (step 8).

Potential solution

The signal reduction with increasing irradiation time is due to peptide assembly and consequent precipitation. In order to quantify the percentage of isomerization in these samples, they can be lyophilized, re-dissolved in H₂O+0.1% TFA and analyzed again by uHPLC-MS.

Problem 5

Solvent leakage between tubing and PDMS chips or between PDMS chips and glass slides (step 12).

Potential solution

Make a wider PDMS chip that can better hold the connection between the tubes and the chip. Another possible solution is not to push the tube to the end of the PDMS hole. In case of leakage between the PDMS chip and the glass slide, improve the bounding by increasing the time of oxygen plasma treatment.

Problem 6

Blocking of a channel due to dirt entering the microfluidic chip (step 12).

Potential solution

Increase the flow rate of the channel where the dirt is accumulated, subtract the solvent inside that channel or apply a stream of air. In case of complicated blockages, discard the chip and use a new one.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to the lead contact to be fulfilled, Juan R. Granja (juanr.granja@usc.es).

Technical contact

Any technical questions should be directed to the technical contact to be fulfilled, Juan R. Granja (juanr.granja@usc.es).

Materials availability

This study did not generate new unique reagents.

Data and code availability

The datasets generated during this protocol are available in our research article published in Chem.¹ Any questions regarding information or data can be addressed to the lead contact, Juan R. Granja (juanr.granja@usc.es).